Role Of SMAD4in The Mechanism Of VPA’s Inhibitory Effect On Prostate Cancer Cell Invasiveness

In recent years, the incidence of prostate cancer (PCa) has risen rapidly. As PCa developed into an advanced stage, a considerable part of cases end up with bone and lung metastases, which accompanies a poor prognosis. Therefore, study on metastatic PCa has become a hot topic in recent years. According to the reports, epithelial-mesenchymal transition (EMT) is an initiation factor of invasiveness and metastasis in most kinds of cancers. TGF-β is a well accepted regulatory factor in EMT, and SMAD4plays a vital role in mediating TGF-β signaling pathway induced EMT. However, there are also some viewpoints holding that SMAD4plays an inhibitory role in PCa’s invasive and metastatic behavior. There is still no conclusion of SMAD4’s effect during PCa’s progression. To summarize the current researches, histone deacetylase inhibitors (HDACIs) exhibit impressive ability in suppressing PCa in many ways, including proliferation, autophagy, angiogenesis and EMT. So far, the relationship between HDACI and SMAD4in PCa’s EMT has not been well reported yet.ObjectiveTo investigate the influence of the histone deacetylase inhibitor (HDACi) valproic acid (VPA) on SMAD4expression and the invasive ability of prostate cancer cell lines.MethodsDU145and PC3cell lines were cultured under5%CO2and37℃; all the experiments were carried out after48h treatment of VPA.1. Transwell assay:DU145and PC3cell lines were treated with0,2.0or5.0mM/L of VPA. After the treatment of VPA, Transwell invasion assay was carried out both in DU145and PC3cell line, then the number of transferred cells was defined with an optical microscope;2. Immunohistochemistry assay:DU145and PC3cell lines were treated with0or5.0mM/L of VPA. After the treatment of VPA, immunohistochemistry assay was carried out both in DU145and PC3cell line, and then FITC combined SMAD4was observed with fluorescence microscope;3. Western blotting:DU145and PC3cell lines were treated with0,2.0or5.0mM/L of VPA. After the treatment of VPA, Western blot was operated both in DU145and PC3cell line, then the expression level of SMAD4and p-AKT were observed with ECL detect system.Results1. Transwell assay:Compared with controls, VPA significantly suppressed invasiveness both in PC3and DU145cells in a dose-dependent way (P<0.01);2. Immunohistochemistry assay:SMAD4was detected predominantly in the cytoplasm of both DU145and PC3cells; and compared to controls, VPA suppressed the fluorescence intensity of SMAD4both in DU145and PC3cells;3. Western blotting:VPA effectively decreased p-AKT’s expression both in DU145and PC3cells in a dose-dependent manner, which consistent with current published researches, suggesting VPA’s onset as an anti-tumor drug; meanwhile, SMAD4’s expression level was also decreased both in DU145(P<0.05) and PC3cells (P<0.01) in a dose-dependent way.ConclusionVPA could impressively suppress the invasive ability of both PC3and Du145cell lines, and meanwhile restrained the expression level of SMAD4, which indicates that VPA may inhibit PCa invasion and metastasis by inhibiting TGF-P induced EMT via restraining SMAD4’s activity. The results may provide more information in clarifying the mechanism of VPA’s anti-invasion capability.